The invention is relative to a molecular genetic method. The copy number of different nucleic acid sequences of a collective of nucleic acid sequences to be investigated, of a so-called nucleic acid sequence collective is compared with the copy number of a different nucleic acid sequence collective with this method. This comparison determines the changes in the copy number of the nucleic acid sequence collectives relative to one another as well as the sites at which these changes occur.
The nucleic acid sequence collectives described here can be in particular genomes or chromosomes or DNA or RNA sequences from different genomes. The further comments, in as far as they refer to genomes, chromosomes or DNA or RNA sequences, are in no way intended to limit the general concept of the nucleic acid sequence collective but rather are used exclusively to simplify the presentation. Of course, that which is disclosed within the framework of the present invention applies in a general manner to any nucleic acid sequence collectives.
The fluorescence marking of nucleic acids and chromosomes and the subsequent fluorescence microscopic detection have been known since 1970 already from publications of Person and Bobrow.
Even the production of in-situ hybridizations using fluorescence markers is described, for example, in U.S. Pat. No. 5,447,841 EP 430,402 A1 and WO 90/05789. This fluorescence in-situ hybridization is used in particular for analyzing numeric and structural chromosomal anomalies. In this method fluorescence-marked DNA sequences are hybridized onto a DNA to be investigated. The marked DNA settles by recombination on the site of the DNA to be investigated which is complementary to the marked DNA. The hybridized, marked DNA sequences can then be detected with fluorescence-microscopic techniques. This detection of fluorescence markers is carried out on metaphase or interphase nuclei as a function of the question posed. For the case in which the localization of the marked DNA sequences on the corresponding chromosome is significant the metaphase cells are investigated. If, however, the number of marked DNA sequences is to be determined, as, e.g., in the analysis of numeric aberrations of chromosomes, it is sufficient to subject the interphase nuclei to fluorescence-microscopic analysis.
A further development of this technique is comparative genomic hybridization, CGH, which is used in particular in the diagnosing of tumors. In contrast to the method cited above, in this method the isolated DNA of a genome to be investigated is marked with fluorescent dyes. This marked DNA is hybridized together with reference DNA on metaphase chromosomes.
Thus, for example, DNA to be investigated which is isolated from tumor tissue (test DNA) is marked in a fluorescent color (e.g. green). This marked test DNA is then hybridized together with normal reference DNA marked in a different color (e.g. red) onto normal metaphase chromosomes which are marked blue, e.g., with 4, 6-diamidino-2-phenylindol (DAPI). During the recombination during the hybridization a competition takes place between the test DNA and the reference DNA for the binding sites on the DNA of the metaphase cell. Normal ranges of the tumor genome will settle with the same probability on the metaphase chromosomes as the corresponding ranges of the reference DNA. These ranges exhibit a certain intensity ratios of the dyes red and green used in the fluorescence-microscopic evaluation. The areas of the tumor genome which exhibit an increase in the copy number of the DNA sequences recombine with a greater probability than the reference DNA with the sites of the metaphase chromosomes complementary to them so that a shift of the intensity ratio in the direction of green occurs at the corresponding sites. On the other hand, the areas of the test DNA in which the copy number of the DNA sequences is reduced are recognized by a red shift of the intensity ratio. The different fluorescence intensities with which the changes in the copy number of the DNA sequences can be recognized are represented via a quantitative evaluation of the green/red intensity ratios as so-called profiles of the ratio values. In this connection a ratio value represents the standardized quotient from the intensities of the green and of the red fluorescence of a certain DNA sequence.
WO 93/18186 describes a method of comparative genome hybridization. In this method the DNA of a genome to be investigated is marked with fluorescent dyes and hybridized on metaphase chromosomes. The intensity of the fluorescence-microscopic signals of the DNA to be investigated is subsequently measured as a function of the position on the metaphase chromosomes. As a result of the comparison of the intensities at different sites of the genome to be investigated which correspond to different DNA sequences conclusions about the existence of such DNA sequences can be drawn which are present relative to the other DNA sequences of the genome with a changed number of copies.
Systematic deviations from the expected ratio values result in this CGH method. Thus, for example, the following factual situation is regularly found for the X chromosome:
The X chromosome is used as an internal control when female tumor DNA (2 X chromosomes) is mixed with male reference DNA (only one X chromosome) and hybridized on metaphase chromosomes. The expected ratio value should be on the average approximately 2.0 due to the double number of X chromosomes in the test DNA. However, in most hybridizations it is distinctly below that value, approximately only between 1.5 and 1.8. In general, a diminution of the dynamics of the CGH ratio values is observed, that is, the ratio values  less than 1 as well as the ratio values  greater than 1 are shifted in direction 1, that is, in the direction of normal value. This dynamic loss results in a reduced sensitivity of the CGH method and rather slight changes in the copy number can no longer be reliably detected since they remain below the significance thresholds. This sharply limits the ability of the CGH results to be interpreted diagnostically, especially if slight variations in the number of copies are involved.
The precise cause of the diminished dynamics of the ratio profiles is not known. A possible explanation for the observed effect might be an insufficient saturation of the non-specific components of the test genomes and reference genomes used. Non-specific components, so-called repetitive sequences, are sites on the genome which comprise frequently occurring DNA sequences and can therefore recombine with various other sequences within the genome. The additive components of this non-specific recombination for test DNA and reference DNA results in a shift of the ratio values in direction 1. During the performance of CGH investigations these repetitive sequences are saturated by non-marked Cot-1-DNA; however, the saturation does not always take place in a complete manner.
The correction of this effect was previously suggested by Lundsteen et al. in that an empirically determined value such as, e.g., 10% is subtracted from the fluorescence intensities measured. However, a significant disadvantage of this empirical correction is the susceptibility to experimental fluctuations. Also, results have shown that such a simple model does not sufficiently describe the observed effects. Karhu et al. show in xe2x80x9cQuality Control of CGH: Impact of Metaphase Chromosomes and the Dynamic Range of Hybridizationxe2x80x9d, Cytometry, 28: pp. 198-205, (1997) that the dynamic range of the ratio values for identical text DNA and reference DNA depends heavily on the ability of the metaphase chromosomes on which the hybridization is carried out to be hybridized and does not correlate with customary checking criteria of the suppression of repetitive sequences such as the hybridization intensity of centromeres.
WO 93/18186 also describes in claims 10, 11 and 17 the quantitative determination of a changed number of copies of DNA sequences of the genome in comparison to those of another genome. To this end the DNA of both genomes is marked in different fluorescent dyes and hybridized onto normal metaphase chromosomes. After the measuring of the intensities as a function of their position on the metaphase chromosomes a profile of the ratio values is formed which reflects the relative intensities of the DNA sequences of the genomes used relative to each other at corresponding sites of the genomes. The ratio profile obtained is standardized using a calibrating sequence known on both genomes in order to be able to subsequently convert the unknown sequences via their ratio values into absolute copy numbers. A correction of the experimentally observed diminution of the dynamics of CGH ratio profiles is not possible in this manner. Even the calibration, described there in claim 12, of changes in copy number via the pair-by-pair comparison of more than two simultaneously hybridized genomes serves solely to convert quotients into absolute values.
The present invention has the problem of indicating a method of comparing the copy number of nucleic acid sequences from different nucleic acid sequence collectives which does not have this diminution of dynamics. Thus, it should be made possible to determine and correct systematic hybridization influences by using an internal standard. Furthermore, the correction should be able to be carried out independently of the nucleic acid sequence collectives to be investigated so that a nucleic acid sequence collective which is completely unknown can be investigated.